The present invention relates to multiple capillary electrophoresis devices.
It is known that conventional gel electrophoresis techniques, in which various samples are injected on a plurality of tracks defined in a gel contained between two plates, are unsatisfactory, given, on the one hand, that they require a certain number of manual operations and, on the other hand, that they do not permit very high migration rates and therefore processing throughputs.
However, major sequencing and genotyping programs require a very high rate of DNA molecule separation and identification.
Electrophoresis techniques are also known which, for migration, use a capillary filled with gel or with another separation matrix having the advantage of being particularly easy to handle, easy to put in and of allowing substantially automatic operation, with higher separation rates than in gel plate electrophoresis by virtue of a strong applicable electric field.
However, the use of a single capillary does not make it possible to achieve the same throughputs as those allowed by electrophoresis techniques using plates which have many tracks in parallel, even though the electric fields that can be applied to a capillary, and therefore the migration rates obtained, are nevertheless higher.
This is why so-called multiple capillary systems have also been proposed, which include a linear array of a plurality of juxtaposed capillaries. In particular, multiple capillary electrophoresis systems have been proposed in which the laser beam for exciting the molecules is directed onto them through the walls of the capillaries, along an axis in the plane of the linear array over which said capillaries are distributed. Said axis is generally perpendicular to the direction in which the capillaries extend, the fluorescence of the molecules being observed using reception means having an optical axis perpendicular to the plane of the linear array of the capillaries.
Reference may, for example, be made in this matter to the publication:
xe2x80x9cA Capillary Array Gel Electrophoresis System Using Multiple Laser Focusing for DNA Sequencingxe2x80x9dxe2x80x94T. Anazawa, S. Takahashi, H. Kambaraxe2x80x94Anal. Chem.xe2x80x94Vol. 68, No. 15,xe2x80x94Aug. 1, 1996xe2x80x94pp. 2699-2704.
However, such a technique is unsatisfactory in view of the detection noise resulting from the interaction between the excitation light and the fluorescence of the walls of the capillary. Further, the laser beam loses intensity as it passes through the capillaries, so that the molecules which lie in the capillaries furthest from the laser source are excited less than those moving through the first capillaries.
In order to solve this problem, it has also been proposed, in particular in:
xe2x80x9cCapillary Array Electrophoresis Using Laser-Excited Confocal Fluorescence Detectionxe2x80x9dxe2x80x94X. Huang, M. Quesada, R. Mathiesxe2x80x94Anal. Chem. 1992, 64, 967-972, to use an excitation beam emitted perpendicularly with respect to the plane of the linear array and to carry out detection of the fluorescence using optical means whose axis coincides with that of the beam, this axis and the excitation beam being moved successively in the course of time from capillary to capillary.
But this technique is not satisfactory either, given that it requires complex mechanical means and, further, the movement of the detection means from one capillary to another leads to a great deal of dead time.
Multiple capillary systems are moreover known in which the molecules passing through the capillaries are excited by laser radiation which is directed, just at the output of the linear array, into the plane of said linear array and perpendicular to the direction in which the capillaries extend. The fluorescence of the molecules excited by this radiation is detected using, in particular, a CCD camera which is oriented with an axis perpendicular to the plane of the linear array of capillaries.
A system of this type is, for example, presented in the publication:
xe2x80x9cAnalysis of Nucleic Acids by Capillary Electrophoresisxe2x80x9dxe2x80x94C. Hellerxe2x80x94pp. 236 to 254 Editions Viewegxe2x80x941997.
Exciting the molecules and detecting the fluorescence outside the capillaries ensures a signal to noise level higher than that accessible in conventional detection layouts, where the excitation and the detection takes place directly through the walls of the capillaries.
However, such a system requires the provision of means preventing the molecules at the output of the various capillaries from diverging too greatly.
This is generally done using laminar buffer flows, which requires, for the cuvette in which the capillaries are accommodated, high-precision mechanical production in glass. In particular, the device will need to be capable of avoiding any gas bubble perturbing the flow.
As will have been understood, such a technique has the major drawback of being very expensive.
Furthermore, it does not allow the operator to handle with ease the capillaries, which require a high degree of alignment precision.
In addition, it requires large volumes of solution which is used for the flows.
What is more, it has been found in practice that this technique is difficult to use except by specialists.
A system for detection at the output of the capillaries without laminar flow, and which avoids these drawbacks, has been proposed by H. Kambara (U.S. Pat. No. 56 67 656 variants 7 and 8). In this system, the detection takes place in the small-dimensioned (0.5 mm) space formed between the electrophoretic separating capillary and a second capillary, which faces the former and is used for passing an electric current. Said detection space is located in an optically favorable buffer-filled cavity. The electric current passes through the detection space between the two capillaries so that the samples migrate from the first capillary to the second. According to the patent of H. Kambara, since the separation between the capillaries is small, these molecules should diverge little in the detection cavity. However, calculations of electrostatics clearly show that, on the contrary, the electric field diverges laterally in the gap between the capillaries. What is more, it found in practice that any system with residual lateral dispersion is inapplicable because it entails resolution losses and contamination between analysis channels. Further, experiments clearly show lateral dispersion of the molecules in the gap between two capillaries, causing loss of analysis resolution. As will have been understood, such a system is not therefore effective. Further, it is difficult to use because it requires precise alignment of capillaries facing one another separated by a small distance.
One object of the invention is therefore to provide a multiple capillary electrophoresis device in which the excitation of the molecules and the detection of their fluorescence are carried out at the output of the capillaries, which does not have the drawbacks of the prior art systems and which is particularly reliable, easy to use, and has a level of performance allowing high-rate sequencing and genotyping.
To that end, the invention provides a multiple capillary electrophoresis device including a plurality of juxtaposed capillaries, means for generating inside the capillaries an electric field which ensures electrophoretic migration, at least one source for emitting a beam intended to excite molecules at the output of the capillary, means for detecting the fluorescence of the molecules excited by said beam, characterized in that it includes means for generating another electric field, referred to as the confinement electric field, which is distributed uniformly around said capillaries and which is substantially parallel to them, this electric field confining the electrophoretic migration field and forcing the molecules to move substantially without diversions in the axis of said capillaries.
Such a device is advantageously supplemented by the various characteristics below, taken individually or in any of their technically feasible combinations:
the device includes at least one intermediate metal electrode directly juxtaposed with the capillaries in the vicinity of their output, as well as a metal electrode which defines the electrophoretic field and which is arranged level with the outputs of the capillaries, parallel to the intermediate electrode and perpendicular to the capillaries, these two electrodes together imposing a confinement electric field at the output of the capillaries;
the potential which is applied to this intermediate electrode is at a value between those of the potentials of the electrodes imposing an electric field between the ends of the capillaries;
it includes a detection cavity in which the capillaries are received at their end, the intermediate electrode being arranged outside this cavity;
the wall of said cavity which faces the intermediate electrode has orifices ensuring electrical contact between said intermediate electrode and the electrode which is arranged level with the outputs of the capillaries;
the capillaries are contiguous, and only one capillary out of two is filled, the electric field at the output of a filled capillary being confined laterally by the confinement electric field at the output of the adjacent unfilled capillaries;
the potential difference applied to the filled capillaries is different from that applied to the unfilled capillaries;
the capillaries are distributed in one or more linear arrays, and in the direction perpendicular to the plane of the linear array(s), the electric field at the output of the capillaries is, further, confined by the geometry of a cavity in which said capillaries are received;
a linear array of capillaries is contained between dielectric plates which, at the output of the capillaries, have a recess and are separated by a distance less than or equal to the external diameter of said capillaries;
it includes, facing the capillaries, a plurality of orifices which are aligned with said capillaries and in which the electric fields at the output of the capillaries in which the molecules migrate are channeled;
the orifices are ends of other capillaries.